UA-Glo® Plus Luminescent Kinase Assay

The UA-Glo Kinase Luminescent Assay Kit enables quantitative measurement of kinase activity in enzymatic experiments. This kit determines kinase activity by quantifying the remaining ATP after the kinase reaction: the amount of residual ATP is inversely correlated with kinase activity. When using this kit, the ATP concentration in the kinase reaction can be up to 10μM; with the UA-Glo Kinase Plus Luminescent Assay Kit, the ATP concentration can reach up to 100μM, and with the UA-Glo Kinase Max Luminescent Assay Kit, the ATP concentration can be as high as 500μM. This kit can detect most kinases, with substrates including peptides, proteins, lipids, or sugars. It is suitable for high-throughput homogeneous screening of kinase inhibitors and can differentiate between ATP-competitive and non-competitive inhibitors by adjusting ATP concentrations.

Product Composition: Luciferase, luciferin, buffer mixture filled in 10 ml or 100 ml brown bottles, specifications as follows:

1. Kinase Reaction
1) Perform the kinase reaction in a 96-well or 384-well white opaque assay plate. The recommended reaction volume is 50 µL for a 96-well plate and 10 µL for a 384-well plate. A concentration gradient of test compounds can be added to the kinase reaction.
2) The concentrations of kinase and substrate should be optimized for different kinases. For the desired signal-to-noise ratio, the kinase concentration within the linear reaction range can be used.
3) ATP concentration: The UA-Glo Kinase Plus luminescence detection kit can accommodate up to 100 µM ATP.
4) The kinase reaction can be carried out in a universal reaction buffer (40 mM Tris-HCl (pH 7.5), 0.1 mg/ml BSA, 20 mM MgCl2) or using a buffer and cofactors reported in the literature.
5) The temperature and duration of the kinase reaction should be set according to the specific kinase. For high-throughput compound screening, it is recommended to optimize the kinase reaction at room temperature (22°C–25°C) to maintain consistent plate temperature during the kinase activity assay.

2. Kinase Activity Assay
1) Remove the UA-Glo Kinase Plus luminescence detection reagent and equilibrate it to room temperature. Gently mix by shaking. The luciferase reaction in the kinase activity luminescence detection reagent is sensitive to temperature changes, so the reagent and assay plate must be equilibrated to room temperature, and the temperature should remain constant (±1°C) during the assay.
2) If the kinase reaction was performed at a non-room temperature, such as 30°C, the assay plate should be equilibrated to room temperature.
3) Add 50 µL of the kinase luminescence detection reagent to a 50 µL 96-well assay plate or 10 µL of the reagent to a 10 µL 384-well assay plate. Mix by shaking and incubate in the dark for 10 minutes.
4) Measure the luminescence signal. Since the luminescence signal is highly stable, the plate can be read within 3 hours after adding the detection reagent if needed.